Many methods for peptide synthesis are described in the literature (for examples, see U.S. Pat. No. 6,015,881; Mergler et al. (1988) Tetrahedron Letters 29: 4005-4008; Mergler et al. (1988) Tetrahedron Letters 29: 4009-4012; Kamber et al. (eds), Peptides, Chemistry and Biology, ESCOM, Leiden (1992) 525-526; Riniker et al. (1993) Tetrahedron Letters 49: 9307-9320; Lloyd-Williams et al. (1993) Tetrahedron Letters 49: 11065-11133; Andersson et al. (2000) Biopolymers 55: 227-250; and Bray, Brian L. (July 2003) Nature Reviews 2: 587-593. The various methods of synthesis are distinguished by the physical state of the phase in which the synthesis takes place, namely liquid phase or solid phase.
Liquid phase methods (often referred to as solution phase methods) of synthesis carry out all reactions in a homogeneous phase. Successive amino acids are coupled in solution until the desired peptide material is formed. During synthesis, successive intermediate peptides are purified by precipitation and/or washes.
In solid phase peptide synthesis (SPPS), a first amino acid or peptide group is bound to an insoluble support, such as a resin. Successive amino acids are added to the first amino acid or peptide group until the peptide material of interest is formed. The product of solid phase synthesis is thus a peptide bound to an insoluble support. Peptides synthesized via SPPS techniques are then cleaved from the resin, and the cleaved peptide is isolated.
In addition to the liquid phase and SPPS techniques described above, a hybrid approach can be utilized. Hybrid synthesis is typically utilized to manufacture complex sequences. For example, in one representative hybrid scheme, complex sequences can be manufactured through the solid phase synthesis of relatively large, protected peptide intermediates, which are subsequently assembled either by solution phase or SPPS methods to produce a final peptide product. Thus, as a step in the synthesis, an intermediate compound is produced that includes each of the amino acid residues located in its desired sequence in the peptide chain with various of these residues having side chain protecting groups. The peptide intermediates are isolated, and the protected peptide intermediates are then coupled in solution to form a complete peptide. See, for example, WO 99/48513.
Peptides can also be manufactured utilizing recombinant techniques, whereby recombinant DNA technologies are utilized in cell-free systems to produce peptides of interest.
Peptides and amino acids from which peptides are synthesized tend to have reactive side groups as well as reactive terminal ends. When synthesizing a peptide, it is important that the amino group of one peptide reacts with the carboxyl group of another peptide. Undesired reactions at side groups or at the wrong terminal end of a reactant produces undesirable by-products, sometimes in significant quantities. These by-products and reactions can seriously impair yield or even ruin the product being synthesized from a practical perspective. To minimize side reactions, it is conventional practice to appropriately mask reactive side groups and terminal ends of reactants to help ensure that the desired reaction occurs.
For example, a typical solid phase synthesis scheme involves attaching a first amino acid or peptide group to a support resin via the carboxyl moiety of the peptide or amino acid. This leaves the amino group of the resin-bound material available to couple with additional amino acids or peptide material. Thus, the carboxyl moiety of the additional amino acid or peptide desirably reacts with the free amino group of the resin-bound material. To avoid side reactions involving the amine group of the additional amino acid or peptide, such amine group is masked with a protecting group during the coupling reaction. Two well-known amine protecting groups are the BOC group and the Fmoc group. Many others have also been described in the literature. After coupling, the protecting group on the N-terminus of the resin-bound peptide can be removed, allowing additional amino acids or peptide material to be added to the growing chain in a similar fashion. In the meantime, reactive side chain groups of the amino acid and peptide reactants, including the resin-bound peptide material as well as the additional material to be added to the growing chain, typically remain masked with side chain protecting groups throughout synthesis.
After synthesis, some or all of the protecting groups can be removed from the peptide product (deprotection). When substantially all protecting groups (terminal protecting groups and side chain protecting groups) are removed, this is referred to as global deprotection. Deprotection can occur contemporaneously with cleaving or can be carried out later if the peptide is to be further processed, modified, coupled to additional peptide or other material, and the like. Some cleaving reagents not only cleave peptide from the support resin, but also cause deprotection to occur at the same time (for example, the strongly acidic cleaving reagents associated with BOC chemistry). Other cleaving reagents are milder than those utilized in BOC chemistry and cleave without causing undue deprotection. The cleaved peptide remains substantially protected after cleaving as a result. The mildly acidic cleaving reagents associated with Fmoc chemistry tend to produce cleaved peptides in a protected state.
Peptide synthesis schemes typically require recovery of peptide material (for example, final peptide product or peptide intermediate) at one or more points during synthesis. For example, in SPPS synthesis methods, peptide material is typically recovered after it has been cleaved from the solid support. Similarly, in solution phase methods, the peptide material is recovered from solution. When peptide intermediates are synthesized and then coupled to produce a final, larger peptide product, as in hybrid approaches, several isolation steps may be required.
Typical methods to recover peptides involve the use of acid/salt chemistry. For example, peptide can be precipitated in aqueous salt (such as sodium chloride), and the solids can then be collected (for example, by vacuum filtration), washed, and dried. However, such methods present problems for commercial scale production, including high impurity levels. The presence of impurities in the solution with a peptide can result in downstream problems as well, if the peptide will be subsequently reacted with other species (such as additional peptides).
Other recovery methods involve concentration of the peptide solution under vacuum, followed by reconstitution with a solvent such as ethanol, methanol or heptane, then precipitation of the peptide by the addition of water. Known reagents used for peptide precipitation include heptane, water, methanol, ethanol, or diethyl ether. Each of these reagents has limitations. For example, some reagents can be extremely flammable, whereas other reagents can have a higher boiling point and therefore require higher temperatures during distillation steps to remove them. Further, the use of some reagents (such as heptane) during precipitation can cause electrostatic charge build up, which limits handling of this reagent. In addition, when the reagent is utilized only in the isolation steps, but not in other processing steps, specific equipment and/or processing steps (for example, to remove the reagent or purge the system) must be dedicated to the isolation steps involving that reagent.
As part of the recovery process, the precipitated peptide is collected, often by passing the composition containing the precipitated peptide through a filter. Characteristics of the precipitated peptide can impact the filterability of the precipitate. For example, the individual peptide particles that make up the peptide precipitate are desirably in a size range that allows for effective filtration of the precipitate. If outside this desirable size range, peptide precipitate can generate fines that clog the filtration apparatus (for example, when particles are too small) or become too tacky and prevent filtration (for example, when particles are too large).
For large-scale production of peptides, issues relating to product recovery and product purity, as well as reagent handling, storage and disposal, can greatly impact the feasibility of the peptide synthesis scheme. Thus, there is a continuing need for peptide synthesis processes capable of producing peptide materials of commercial interest in large batch quantities. Recovery of peptide material after synthesis, for example, by precipitation, is one aspect of the synthesis in which improvement is needed. Conventional methodologies may result in impurity levels that are higher than desirable.